on Radish (Raphanus sativus L.) Root β-amylase

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1 Journal of Stress Physiology & Biochemistry, Vol. 8 No , pp ISSN Original Text Copyright 2012 by Sarowar Jahan, Shaela Pervin, Shariar Shovon, Dev Sharma Roy and Habibur Rahman ORIGINAL ARTICLE Effect of Metal Ions, Chelating Agent and SH-Reagents on Radish (Raphanus sativus L.) Root β-amylase Sarowar Jahan M.G.*, M. Shaela Pervin, M. Shariar Shovon, S.C. Dev Sharma, Narayan Roy, M. Habibur Rahman Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh Telephone: ; Fax: * sjahan.biochem@ru.ac.bd Received May Metal ions play vital roles in enzymes. They may also show sensitivity to various sulfhydryl reagents and chelating reagents. Effect of some metal ions, EDTA and sulfhydryl reagents on the activity of partially purified β-amylase of radish root were studied. Amylolytic activity of purified enzyme was increased substantially in the presence of Ca 2+, Mg 2+, and Zn 2+. Some other divalent cations Cu 2+, Pb 2+, Sn 2+, and Hg 2+ almost completely ceased the enzyme activity. Cobalt (II), Manganese (II), and Iron (III) exhibited moderate activating effects on the activity. Of the monovalent cations, Na + and Ag + reduced the β-amylase activity, while K + increased. The chelating agent EDTA was found to be effective in the enzyme. Sulfhydryl reagents, Iodoacetic acid and N-Ethylmaleimide showed marginal inhibitory effect, but p-hydroxymercuribenzoic acid (PCMB) almost completely stopped the enzyme activity. The addition of thiol compounds such as cysteine could reverse the inhibitory effect of heavy metals and PCMB. The results indicate that sulfhydryl groups of radish root β-amylase were essential for the activity although it is not clear whether the sulfhydryl groups were directly involved in catalysis. Key words: β-amylase / chelating agent / metal ions / radish / sulfhydryl reagent

2 181 Effect of Metal Ions... ORIGINAL ARTICLE Effect of Metal Ions, Chelating Agent and SH-Reagents on Radish (Raphanus sativus L.) Root β-amylase Sarowar Jahan M.G.*, M. Shaela Pervin, M. Shariar Shovon, S.C. Dev Sharma, Narayan Roy, M. Habibur Rahman Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh Telephone: ; Fax: * sjahan.biochem@ru.ac.bd Received May Metal ions play vital roles in enzymes. They may also show sensitivity to various sulfhydryl reagents and chelating reagents. Effect of some metal ions, EDTA and sulfhydryl reagents on the activity of partially purified β-amylase of radish root were studied. Amylolytic activity of purified enzyme was increased substantially in the presence of Ca 2+, Mg 2+, and Zn 2+. Some other divalent cations Cu 2+, Pb 2+, Sn 2+, and Hg 2+ almost completely ceased the enzyme activity. Cobalt (II), Manganese (II), and Iron (III) exhibited moderate activating effects on the activity. Of the monovalent cations, Na + and Ag + reduced the β-amylase activity, while K + increased. The chelating agent EDTA was found to be effective in the enzyme. Sulfhydryl reagents, Iodoacetic acid and N-Ethylmaleimide showed marginal inhibitory effect, but p-hydroxymercuribenzoic acid (PCMB) almost completely stopped the enzyme activity. The addition of thiol compounds such as cysteine could reverse the inhibitory effect of heavy metals and PCMB. The results indicate that sulfhydryl groups of radish root β-amylase were essential for the activity although it is not clear whether the sulfhydryl groups were directly involved in catalysis. Key words: β-amylase / chelating agent / metal ions / radish / sulfhydryl reagent β-amylase is an important enzyme used in the food processing, brewing and distil industries (Priest, 1984). Its main form of introduction is in the preparation of syrups from starch via saccharification processes (Boivin, 1997). The practical interest of β-amylase centres on its capacity to produce maltose syrup from starch (Boivin, 1997; Dicko et al., 2000). This maltose containing syrups can be used as moisture conditioners, crystallisation inhibitor, stabilizer, carriers, and bulking agents (Badal et al., 1989). The extensive application of amylases in the food, starch liquefaction and saccharification, detergent, textile, paper, brewing and distilling industries has paved a way for their large-scale commercial production (Gupta et al., 2003). With the advent of

3 Sarowar Jahan et al 182 new frontiers in biotechnology, the spectrum of nucleophilicity and catalytic activity (Bardwell, amylase application has expanded into many other 2005; Giles et al., 2003). fields, such as clinical, medical and analytical In order to obtain an insight into these, an chemistries. attempt has been made to determine the effect of Many substances alter the activity of an enzyme various metal ions and the chelating reagent EDTA by combining it in a way that influences the binding on the radish root β-amylase activity. Besides, the of the substrate (Dogan et al., 2007). These possible effects of sulfhydryl reagents (phydroxymercuribenzoic acid, N-Ethylmaleimide and substances can be called as effectors either being activators or inhibitors. Metal ions can be Iodoacetic acid) on amylase activity have also been considered as good examples, different metals investigated. exhibiting different behaviors in their ability to act MATERIALS AND METHODS as effectors. Li et al., (2005) also stated that metal Materials binding to enzymes plays an important role in their Mature, healthy Raphanus sativus (radish) roots activation and stabilization. Most amylases are were collected directly from the cultivation field known to be metal ion dependent enzymes (Pandey during the winter season and these roots were et al., 2000; Gupta et al., 2003; Ramachandran et used. Sephadex G-150 was from Pharmacia Fine al., 2004). According to Leveque et al., (2000), a Chemicals Uppsala, Sweden. Soluble potato starch metal ion present at high concentrations might and Bovin serum albumin (BSA) were obtained from compete with another metal present at a lower Sigma Co. p-hydroxymercuribenzoic acid (PCMB), N- concentration and replace it at a metal-binding site, Ethylmaleimide and Iodoacetic acids were even if its affinity for the binding site is lower. An purchased from Fluka. All others chemicals were of accepted property of amylases is their content of analytical grade and also purchased from Sigma Ca 2+ as an integral part of the enzyme molecule and Inc., USA. their consequent activation by Ca 2+ and inhibition Preparation of the crude extract by chelating reagents (Mar et al., 2003; Kiran and Fresh, mature and healthy radish roots were Chandra, 2008). Besides, amylases have been washed thoroughly with distilled H reported to be inhibited by heavy metal ions such 2 O, cut into small as Ag+, Hg 2+, Cu 2+, Fe 2+ pieces and ground with distilled H, etc., suggesting the 2 O and sand in a morter at 4 involvement of cysteine residue in enzyme activity. o C. The suspension was then filtered through few layers of cheesecloth in a cold room. The importance of cysteine residues for catalysis The filtrate was clarified further by centrifugation at has been described for amylases independent of 10,000 g for 15 min at 4 their origin (Pandey et al., 2000; Lo et al., 2001; Diaz o C. The supernatant was then dialyzed against distilled H et al., 2003). The thiol of a cysteine residue 2 O for 48h at 4 o C and centrifuged at 10,000 g for 15 min at 4 occasionally acts as a ligand of metal ions (Tatara et o C. The clear supernatant obtained was used as crude al., 2005). Amylases are also found sensitive to enzyme extract. various sulfhydryl modifying reagents as free thiol or sulfhydryl groups are considered to be useful in stability, redox behaviour, metal binding, acidity,

4 183 Effect of Metal Ions... Purification of β-amylase All steps of purification were carried out at 4 0 C. The enzyme was purified to homogeneity by the sequential steps of acetone precipitation, followed by DEAE- cellulose chromatography and gel filtration on Sephadex G-150. Enzyme precipitation The crude extract was initially fractioned by 50% (v/v) chilled (-20 o C) acetone saturation. After centrifugation at 10,000 g for 15 min, the precipitated pellets were collected and resuspended in two-pellet volume of cold buffer. The solution was dialyzed against 10 mm phosphate buffer of ph 7.0 for overnight. Undissolved particles were removed by centrifugation and the clear solution was used for further purification. DEAE- cellulose chromatography The enzyme preparation obtained from the previous step was passed over a DEAE-cellulose column (20.0 X 1.0 cm) previously equilibrated with 10 mm phosphate buffer of ph 7.0 at 4 o C. The buffer was first passed through the column and then the enzyme was eluted with the same buffer containing a linear gradient of NaCl (0-500 mm) at a flow rate of 30 ml/h. Fraction with β-amylase activity was pooled and dialyzed against 10 mm phosphate buffer of ph 7.0 at 4 o C and concentrated by Millipore. Gel filtration chromatography The dialyzed, concentrated enzyme preparation resulting from the anion exchange chromatography was applied to a column of Sephadex G-150 (150 X 3.0 cm) preequilibrated with 10 mm phosphate buffer of ph 7.0 at 4 o C. The protein was eluted with the same buffer at a flow rate of 15 ml/h. Enzymatically active fraction from gel filtration step was pooled separately, concentrated by commercial sucrose and applied for molecular weight determination by gel filtration and electrophoresis. Enzyme assay One milliliter of enzyme was added to 1 ml of 1% soluble starch containing 0.1 M phosphate buffer ph of 6.7 and the mixture was incubated at 37 o C for 15 min. The amount of reducing sugar produced was determined by Somogyi (1952) and Nelson (1944) methods. One unit of enzyme activity was defined as the amount; which catalyzed the formation of 1 μmol of maltose under the assay conditions. Protein concentration was determined by Lowry s phenol method (1951), using crystalline BSA as the standard. SDS-polyacrylamide gel electrophoresis (SDS- PAGE) SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (1970). The PAGE was performed with 7% gels at 110 V and 30 ma. The protein bands on the gel was dyed by using of 0.25% Coomassie brilliant blue R- 25 (CBB) solution containing 50 % methanol and 10 % acetic acid. Effect of metal ions, EDTA and sulfhydryl reagents on β-amylase activity The effect of various metal ions, chelating agent and inhibitors on the activity on β-amylase was examined by incubating the enzyme with substrate solution (comprising of 1% starch in 0.1 M phosphate buffer ph 6.7) at 37 o C in the presence of the respective ions or chemicals for 5 min and at the end of incubation period, aliquots were withdrawn and assayed. In all of the above cases,

5 Sarowar Jahan et al 184 the β-amylase activity is expressed as a percentage the enzyme with Na + and Ag + reduced the activity. of the control enzyme activity (100%). Divalent cations like Ca 2+, Mg 2+, and Zn 2+ increased the activity of the purified β-amylase. Usually, the RESULTS AND DISCUSSION role of Ca 2+ in maintaining the stability and Purification of radish root β-amylase structure of the amylase is well documented The crude enzyme solution was precipitated by (Parkin, 1993). Similar to other plant β-amylases, the addition of water-miscible organic solvent, the radish root β-amylase was severely inhibited by acetone. After centrifugation, the concentrated Cu 2+, Pb 2+, Sn 2+, and Hg 2+. Notably Fe 2+ moderately dialyzed enzyme solution was passed over a column reduced the amylolytic activity. of DEAE-cellulose for separation on the basis of Effect of EDTA on radish root β-amylase activity ionic property of β-amylase. The active fraction It is well known that amylases contain Ca 2+ as an obtained from the ion exchange chromatography essential component of the enzyme molecule and was applied to a column of Sephadex G-150 (50 are often inhibited by the chelating reagent EDTA X1.0 cm) for further separation. The molecular (Mar et al., 2008; Kiran and Chandra, 2008) weight of radish root β-amylase, determined from presumably because of its chelating properties. the experiment with Sephadex G-150 gel filtration Interestingly, radish root β-amylase increased column was 62 kda (Data not shown). This was in activity with the addition of EDTA (Table 3). good agreement with the molecular weight (61 kda) determined by SDS-PAGE (Data not shown). Effect of sulfhydryl reagents on radish root β- Hence, radish root β-amylase was a monomer. The amylase activity present value (61 kda) was very close to the The effect of the sulfhydryl reagents (phydroxymercuribenzoic acid, N-Ethylmaleimide and molecular weights of β-amylase isolated from yam tuber (60 kda) and ginseng (63 kda) reported by lodoacetic acid) on β-amylase activity are presented Arai et al., (1991) and Yamasaki et al.,(1989), in Table 3. The minimum amount of these reagents respectively. Larger multimeric β-amylases have inducing a detectable inhibition of activity in radish been reported from leaves of potato (111 kda) root β-amylase amylase was found to be 0.1 mm. (Vikso-Nelson et al., 1997) and potato tubers (122 From the above results, it was found that kda) (Yamasaki et al., 1989). Iodoacetic acid and N-Ethylmaleimide reduced the The effect of metal ions on radish root β-amylase activity of β-amylase moderately. The enzyme was In the enzyme action, metallic cofactors are almost completely inhibited by the presence of important because their presence or absence heavy metals such as Cu 2+, Pb 2+, Sn 2+ Hg 2+ and by SHinhibitor such as PCMB, the activity being restored regulates enzyme activity. The presence of specific metallic ions along with food content can inhibit or by thiol compounds such as cysteine. The result enhance amylase activity, and therefore the rate of indicates that an SH group exits in the molecular digestion. Various metal ions were tested for structure, as in other plant β-amylase. The activation/inhibition effects that were shown in inactivation caused by PCMB was similar to that Table 2. Among monovalent cations K + increased found in other plant β-amylases (Marshall, 1975). the enzyme activity substantially, but incubation Plant β-amylase has been reported to require free

6 185 Effect of Metal Ions... sulfhydrylgroups for activity and is inhibited by heavy metals as well as other binding reagents (Thoma et al., 1971; Trachuk and Tipples, 1966). Table 1: Summary of radish root β-amylase purification Purification Step Total Protein (mg) Total Activity (unit) Specific Activity (unit/mg) Yield Purification (fold) Crude Extract Acetone Precipitation & Dialysis DEAE-cellulose Sephadex G Table 2: Effects of various metal ions on radish root β-amylase Metal Ions Concentration Residual Activity Induction Inhibition (mm) Control NaCl KCl AgNO CaCl MgCl ZnCl MnCl CoCl FeCl CuCl PbCl SnCl HgCl FeCl Table 3: Effects of chelating reagent EDTA and sulfhydryl reagents on radish β-amylase Chemical Reagent Concentration (mm) Residual Activity Induction Inhibition Control EDTA *PCMB Iodoacetic acid N-Ethylmaleimide * p-hydroxymercuribenzoic acid

7 Sarowar Jahan et al 186 Protein Enzyme Absorbance at 280 nm Linear gradient of NaCl Specific activity (U/mg) Fraction No. (3ml/tube) Figure 1: Elution profile of radish root β-amylase on DEAE-cellulose column. The enzyme solution (485.5mg protein) obtained after acetone precipitation and dialysis was applied to the column previously equilibrated with 10 mm phosphate buffer ph 7.0 at 4 0 C eluted with the same buffer containing a linear gradient of NaCl (0-500 mm) Protein 0.4 Enzyme F-1c 0.4 Absorbance at 280 nm F-1a F-1b Specific activity (U/mg) Fraction No. (3 ml/tube) 0 Figure 2: Elution profile of F-1 fraction on Sephadex G-150 column. Protein (59.4 mg) was applied to column pre-equilibrated with 10 mm phosphate buffer ph 7.0 at 4 0 C. The protein was eluted with the same buffer with same ph.

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